Atomic force microscopy, also called scanning force microscopy and scanning probe microscopy, has become an important tool for biological science, with significant application to imaging samples such DNA and living cells in solution. When imaging biological samples, an atomic force microscope (AFM) is usually operated with a liquid cell, because the samples need to remain immersed in order to retain natural characteristics.
Generally atomic force microscopes for biological science employ liquid cell housings that contain a specimen in fluid. A fluid cell for an atomic force microscope is described by Hansma et al. in “Atomic Force Microscope with Optional Replaceable Fluid Cell,” U.S. Pat. No. 4,935,634 issued Jun. 19, 1990. The optional and replaceable probe-carrying module includes the provision for forming a fluid cell around the AFM probe.
An electro-chemical liquid cell for use with an atomic force or scanning tunneling microscope is described by Lindsay et al. in “Scanning Probe Microscope for Use in Fluids,” U.S. Pat. No. 5,750,989 issued May 12, 1998. A hermetically sealed chamber may be formed around a sample by a seal between the scanner of the microscope and its frame.
Knauss et al. describes a hyperbaric hydrothermal atomic force microscope with a gas pressurized microscope base chamber and a sample cell environment in “Hyperbaric Hydrothermal Atomic Force Microscope,” U.S. Pat. No. 6,437,328 issued Aug. 20, 2002. The AFM images solid surfaces in liquid or gas that flow within the sample cell at pressures greater than normal atmospheric pressure.
Tapping mode AFM has become an important tool, capable of nanometer-scale resolution on biological samples. The periodic contact with the sample surface minimizes frictional forces, avoiding significant damage to fragile or loosely attached samples. A representative tapping AFM is described by Elings et al. in “Tapping Atomic Force Microscope,” U.S. Pat. No. 5,412,980 issued May 9, 1995.
Liquid tapping-mode AFM, also referred to as liquid cyclic-mode AFM, is a scanning probe imaging mode that is suitable for biological imaging and is used frequently to obtain nanometer-scale resolution on fragile specimens. Liquid tapping-mode AFM helps to minimize friction damage that is characteristic of contact-mode AFM, to reduce van der Waals forces, and to eliminate capillary forces between an AFM cantilever tip and a specimen. Tapping mode has been used to image DNA in situ, the folding and unfolding of individual titin molecules, crystal growth, Langmuir-Blodgett films, polymers, living plant cells, red and white blood cells, moving myosin V molecules, and numerous other biological samples.
AFMs in a liquid tapping mode typically employ a cantilever, an external piezoelectric oscillator, and an optical displacement-sensing component. An AFM operating in a vibrating, cyclic or tapping mode may use a piezoelectrically actuated microcantilevered probe. Typically, the probe is a micro-electrical-mechanical-system (MEMS) device, micromachined from bulk silicon and silicon-on-insulator (SOI) wafers with a piezoelectric film patterned along a portion of the microcantilever. At the free end of the cantilever is a tip with nanometer-scale radius, optimally shaped to probe the sample surface. The microcantilever is displaced by voltage applied to the piezoelectric actuator, resulting in a controlled vertical movement of the tip. Control electronics drive the microcantilever while simultaneously positioning it vertically to track the sample topography and follow the surface features. A macro-scale position actuator such as a piezotube may be used to null the position of the cantilever, following the topology of the sample as the probe is scanned over the surface. Smaller AFM cantilevers have been developed, contributing to improvements in the imaging speed of the liquid tapping mode.
Xu et al. describes an AFM with a cantilever tip for probing a biological specimen in “Atomic Force Microscope for Biological Specimens, U.S. Pat. No. 5,874,668 issued Feb. 23, 1999. The cantilever is designed to identify physiologically and pharmacologically important biomolecules and their constituent subunits. For example, a cantilever can be manufactured to be biospecific, allowing the identification of specific voltage-sensitive tissues and biomolecules.
A sensor using a cantilever to detect a selected target species is disclosed by Lee et al. in “Chemical and Biological Sensor Using an Ultra-Sensitive Force Transducer,” U.S. Pat. No. 5,807,758 issued Sep. 15, 1998. This chemical and biological sensor has a cantilever with attached chemical modifiers capable of undergoing a selective binding interaction. A target specimen in contact with the cantilever can generate an electric or magnetic field that induces a measurable deflection. The target molecule may be in liquid phase or in vapor phase.
Another chemical sensor using microcantilevers is described by Thundat in “Microcantilever Detector for Explosives,” U.S. Pat. No. 5,918,263 issued Jun. 29, 1999. This apparatus detects explosive vapor phase chemical, employing a cantilever and a heater for increasing the surface temperature of the cantilever that causes combustion of the adsorbed explosive vapor phase chemical. The combustion results in a deflection and a resonance response of the cantilever.
A magnetically modulated cantilever is described by Han et al. in “Magnetically-Oscillated Probe Microscope for Operation in Liquids,” U.S. Pat. No. 5,753,814 issued May 19, 1998. The invention employs an AC-driven atomic force microscope with a ferrite-core solenoid for modulating the magnetic cantilever. The detection system for the magnetically modulated AC-AFM incorporates AC coupling of the signal from the position sensitive detector/beam deflection detector in order to remove the DC component of the signal. The result is an improved dynamic range over systems using DC coupling.
Attempts have been made to increase the speed of AFM imaging. An amplitude detection circuit is used to dynamically control the cantilever drive signal in an amplitude domain, as described by Adderton et al. in “Dynamic Activation for an Atomic Force Microscope and Method of Use Thereof,” U.S. Patent Application 2002/0062684, published May 30, 2002.
Lee et al. have used piezoelectric lead-zirconate-titanate (PZT) actuated cantilevers to achieve 1,030 pixels/s and tip speeds of 16 μm/s, as disclosed in J. Vac. Sci. & Tech., B 15(4), 1559 (1997). Cantilever probes with a thin integrated film of zinc oxide (ZnO) serving as an actuator have achieved a resonance frequency on the order of 15 kHz in liquid. The results are faster imaging speeds and improved tuning capability, as reported by Sulchek et al. in Rev. Sci. Instrum. 71(5), 2097 (2000), and by Rogers et al. in Rev. Sci. Instrum. 73(9), 3242 (2002). For most biological samples, conventional AFMs can scan at speeds of a few tens of microns per second, which could require several minutes to produce a 512×512 pixel image. Thus, the scan rate of an AFM may be too slow for applications where biological and chemical processes occur in less than a minute.
Faster measurement times would help shrink the existing separation between the time scales of force spectroscopy experiments and the time scales of molecular dynamics calculations. Quicker scan speeds would reduce the time spent locating interesting features and would enable the study of dynamics occurring in liquid or physiological environments. An improved method for scanning a specimen that is in a liquid environment would scan more quickly and provide better tuning capability than currently used AFM cantilever probes. In addition, an improved method would provide a real-time imaging tool for studying dynamic phenomena in physiological conditions.
Therefore, what is needed is a structure and a method for quicker imaging times for contact and tapping mode atomic force microscopy in liquid, and for sensing target chemical and biological species in liquid, overcoming the deficiencies and obstacles described above.